Cell membranes include a number of channels through which molecules such as nutrients, waste products, ions, and small molecules pass. Ion channels are one example of membrane channels that are found in the membrane of all cells. Typical ion channels are composed of a trans-membrane protein or group of proteins that selectively allow the passage of ions through the membrane lipid bilayer. Named after the predominant ion passing through the channel, examples of ion channels include sodium (Na+) channels, potassium (K+) channels, chloride (Cl−) channels, and calcium (Ca2+) channels. An ion channel can be permanently open, such as is found in a potassium leak channel. An ion channel can also be voltage gated, and one example is the sodium channel. Alternatively, ion channels can be ligand gated. Ligand gated channels are ion channels whose permeability to ions is sensitive to the binding of a specific ligand, such as a neurotransmitter. Examples of neurotransmitters include, but are not limited to, acetylcholine, glutamate, glycine, or γ-aminobutyric acid. The category of membrane channels can also include membrane channels that are not ion-conducting, but rather permit passage of other molecules into and/or out of the cell.
The structures of ion channels differ among channel families. Each channel includes various protein subunits encoded by different genes that can be selectively expressed in certain cell types or during certain periods of development and growth of the organism.
Ion channels play a critical role in shaping the electrical activity of neuronal and muscle cells, as well as controlling the secretion of neurotransmitters and hormones. Because of their relevance to a variety of physiological processes in vertebrates, such as muscle contraction, insulin release from the pancreas, and neurotransmitter release in the nervous system, ion channel research has gained increasing importance to the pharmaceutical industry.
In particular, calcium channels are important targets for further research because they are ubiquitous ion channels that specifically mediate Ca2+ passage into and out of cells. Ca2+ entering the cell through voltage-gated Ca2+ channels serves as the second messenger of electrical signaling: initiating events such as contraction, secretion, synaptic transmission, and gene expression. Calcium channels can be categorized as L-type (for long lasting), T-type (for transient), N-type (for neither L-type nor T-type, or for neuronal), P-type (for Purkinje cell), Q-type, and R-type (for resistant) based upon factors such as activation and inactivation kinetics, ion specificity, and sensitivity to drugs and toxins. Except for the T-type channel, which is a low voltage activated (LVA) channel, the L-, N-, P-, Q-, and R-types are high voltage activated (HVA). In other words, the HVA channels exhibit activation thresholds that are normally above −40 mV. Although L-type and T-type Ca2+ currents are recorded in a wide range of cell types, N-, P-, Q-, and R-type currents are most prominent in neurons.
Design of an assay for identifying a new drug that has specific modulatory effects on an ion channel is hindered by the fact that the ion channel is made of multiple protein subunits, and the function of the ion channel can be evaluated only when the channel is within a membrane lipid bilayer. In particular, a compound identification assay for calcium channel modulation is complicated by such factors as: 1) native channel expression at low density; 2) difficulty to clamp by classical voltage-clamp methods (many ion channels that are candidates for study occupy cells that are difficult to clamp, and examples of these cells include dendrites, nerve terminals, and muscle cells due to their complex infoldings); and 3) the small current of ion channels tends to be masked by those of many other channels.
Existing technologies for identifying ion channel modulators are a compromise between throughput, physiological relevance, sensitivity and robustness. A widely accepted assay for studying ion channel function is the patch-clamp technique (Neher (1992), Neuron, 8:605-612). Although several companies are attempting to automate the patch-clamp process, the current complexity and reproducibility of the experimental setup currently renders it unsuitable for a high throughput screen (HTS) application. Optical recording using voltage sensitive dyes or radioisotopes has become popular for the study of voltage-gated ion channels, including calcium channels. Such methods are more readily automated and give much higher throughput for drug screening applications (currently, up to 100,000 compounds per day), and may provide a more sensitive measure of ion channel activity (Xu, et al. (2001), Drug Discovery Today, 6:1278-12887). However these assays frequently require pharmacologic intervention to activate the channels under investigation, leading to the possibility of generating false positive results (Denyer, et al. (1998), Drug Discovery Today, 3:323-332).
Another assay technique adaptable for HTS is the competitive binding assay. Typically, a vacuum filtration method is used to quantify the binding of a radio-labeled specific ligand to an ion channel. The ligand bound to the channel remains on the filter and the unbound free ligand is washed out with wash buffer. This procedure can be cumbersome because it takes a relatively long time and requires large volumes of wash buffer. Further, the separation procedures involve repeatedly washing with the wash buffer, thereby generating a large volume of radioactive liquid waste, which is not only expensive to dispose of, but also presents a potential health hazard to persons performing the experiment.